As a master regulator of cellular stress response, p53 plays an important role in cell fate decisions. The activity of p53 therefore must be precisely regulated. Among numerous proteins involved in p53 control, MDM2 and MDMX are the key players, as evidenced by the fact that knockout of either mdm2 or mdmx in mice resulted in p53-dependent lethality. However, little is known about why both MDM2 and MDMX are required in p53 control, and the molecular mechanisms underlying MDM2 and MDMX-mediated p53 regulation remain not fully defined. Others and we have shown that MDM2 and MDMX form a heterocomplex and, more importantly, that they depend on each other in p53 regulation. Structural and biochemical evidence further indicate that formation of the MDM2/MDMX heterocomplex is favored. Our hypothesis is that the heterocomplex represents the physiological form of MDM2 and MDMX in p53 control. The proposed studies will use mouse models to directly test this hypothesis. We will also fully characterize the MDM2/MDMX heterocomplex in p53 ubiquitination and in regulation of the p53 response to stress. The specific aims are: 1) using animal models to examine a role of the MDM2/MDMX complex in p53 regulation;2) characterize the MDM2/MDMX complex-mediated p53 ubiquitination;3) investigate cellular mechanisms that regulate the MDM2/MDMX complex. With the knock-in mice being successfully generated, we are very favorably positioned to carry out the proposed studies. The findings obtained from the proposed work are expected not only to shed light on the non-redundant function of MDM2 and MDMX, but also to provide a novel mechanism of p53 regulation. PUBLIC HEALTH RELEVANCE: This application aims to directly test the importance of the MDM2/MDMX heterocomplex in p53 control by using animal models. In parallel, the MDM2/MDMX complex-mediated p53 ubiquitination will be fully characterized and cellular mechanisms of MDM2/MDMX regulation will be investigated. Through these lines of investigation, we will test the hypothesis that the MDM2/MDMX heterocomplex is the physiological E3 ligase for p53, which, if proven, will not only represent a new paradigm of p53 regulation but also shed lights on the non-redundant function of MDM2 and MDMX. In addition, the pivotal role of the MDM2/MDMX complex in p53 control presents an ideal target for p53 activation, e.g. disassociation of the heterocomplex, which may have therapeutic implications.